EP0817785B1 - Substrates for beta-lactamase and uses thereof - Google Patents

Substrates for beta-lactamase and uses thereof Download PDF

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EP0817785B1
EP0817785B1 EP96912454A EP96912454A EP0817785B1 EP 0817785 B1 EP0817785 B1 EP 0817785B1 EP 96912454 A EP96912454 A EP 96912454A EP 96912454 A EP96912454 A EP 96912454A EP 0817785 B1 EP0817785 B1 EP 0817785B1
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group
compound
lactamase
cell
alkyl
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EP0817785A2 (en
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Roger Y. Tsien
Gregor Zlokarnik
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University of California
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    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • C07D501/14—Compounds having a nitrogen atom directly attached in position 7
    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
    • C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
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    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
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    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2415/00—Assays, e.g. immunoassays or enzyme assays, involving penicillins or cephalosporins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers

Definitions

  • the present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to compositions and methods for use in measuring gene expression.
  • a reporter gene assay measures the activity of a gene's promoter. It takes advantage of molecular biology techniques, which allow one to put heterologous genes under the control of any promoter and introduce the construct into the genome of a mammalian cell [Gorman, C.M. et al., Mol. Cell Biol. 2 : 1044-1051 (1982); Alam, J. and Cook, J.L., Anal.Biochem. 188: 245-254, (1990)]. Activation of the promoter induces the reporter gene as well as or instead of the endogenous gene. By design the reporter gene codes for a protein that can easily be detected and measured. Commonly it is an enzyme that converts a commercially available substrate into a product. This conversion is conveniently followed by either chromatography or direct optical measurement and allows for the quantification of the amount of enzyme produced.
  • Reporter genes are commercially available on a variety of plasmids for the study of gene regulation in a large variety of organisms [Alam and Cook, supra ]. Promoters of interest can be inserted into multiple cloning sites provided for this purpose in front of the reporter gene on the plasmid [Rosenthal, N., Methods Enzymol. 152: 704-720 (1987); Shiau, A. and Smith, J.M., Gene 67 : 295-299 (1988)]. Standard techniques are used to introduce these genes into a cell type or whole organism [e.g., as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Expression of cloned genes in cultured mammalian cells. In: Molecular Cloning, edited by Nolan, C. New York: Cold Spring Harbor Laboratory Press, 1989]. Resistance markers provided on the plasmid can then be used to select for successfully transfected cells.
  • the reporter gene under the control of the promoter of interest is transfected into cells, either transiently or stably.
  • Receptor activation leads to a change in enzyme levels via transcriptional and translational events.
  • the amount of enzyme present can be measured via its enzymatic action on a substrate.
  • the substrate is a small uncharged molecule that, when added to the extracellular solution, can penetrate the plasma membrane to encounter the enzyme.
  • a charged molecule can also be employed, but the charges need to be masked by groups that will be cleaved by endogenous cellular enzymes (e.g., esters cleaved by cytoplasmic esterases).
  • the use of substrates which exhibit changes in their fluorescence spectra upon interaction with an enzyme are particularly desirable.
  • the fluorogenic substrate is converted to a fluorescent product.
  • the fluorescent substrate changes fluorescence properties upon conversion at the reporter enzyme.
  • the product should be very fluorescent to obtain maximal signal, and very polar, to stay trapped inside the cell.
  • the signal terminates with the bleaching of the fluorophore [Tsien, R.Y. and Waggoner, A.S. Fluorophores for confocal microscopy: Photophysics and photochemistry. In: Handbook of Biological Confocal Microscopy, edited by Pawley, J.B. Plenum Publishing Corporation, 1990, pp. 169-178]. These numbers illustrate the theoretical magnitude of signal obtainable in this type of measurement. In practice a minute fraction of the photons generated will be detected, but this holds true for fluorescence, bioluminescence or chemiluminescence. A good fluorogenic substrate for a reporter enzyme has to have a high turnover at the enzyme in addition to good optical properties such as high extinction and high fluorescence quantum yield.
  • the membrane-permeant compounds may be transformed into substantially membrane-impermeant compounds.
  • Another object of the invention is to provide ⁇ -lactamase reporter genes.
  • a further object of the present invention is to create cells containing the ⁇ -lactamase reporter genes functionally linked to a promotor such that when the promotor is turned on, the reporter gene will be expressed. Expression of the ⁇ -lactamase is measured with the ⁇ -lactamase substrates which emit light after hydrolysis by the ⁇ -lactamase.
  • a further object of the invention is to use the ⁇ -lactamase reporter genes in cells and the ⁇ -lactamase substrate compounds of the present invention to screen for biochemical activity.
  • ⁇ -lactamase substrates are provided of the formula (I) exhibiting fluorescence resonance energy transfer (“FRET") when excited, wherein:
  • this invention provides methods for determining whether a sample-contains ⁇ -lactamase activity.
  • the methods involve contacting the sample with a compound substrate of the invention, which exhibits fluorescence resonance energy transfer when the compound is excited; exciting the compound; and determining the degree of fluorescence resonance energy transfer in the sample.
  • a degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of ⁇ -lactamase activity.
  • One embodiment of this method is for determining the amount of an enzyme in a sample.
  • determining the degree of fluorescence resonance energy transfer in the sample comprises determining the degree at a first and second time after contacting the sample with the substrate, and determining the difference in the degree of fluorescence resonance energy transfer. The difference in the degree of fluorescence resonance energy transfer reflects the amount of enzyme in the sample.
  • this invention provides methods for determining the amount of ⁇ -lactamase activity in a cell.
  • the methods involve providing a host cell transfected with a recombinant nucleic acid molecule comprising expression control sequences operatively linked to nucleic acid sequence; coding for the expression of a ⁇ -lactamase; contacting a sample comprising the cell or an extract of the cell with a substrate for ⁇ -lactamase of the invention; and determining the amount of substrate cleaved, whereby the amount of substrate cleaved is related to the amount of ⁇ -lactamase activity.
  • this-invention provides methods for monitoring the expression of a gene operably linked to a set of expression control sequences.
  • the methods involve providing a host cell transfected with a recombinant nucleic acid molecule comprising expression control sequences operatively linked to nucleic acid sequences coding for the expression of a ⁇ -lactamase, except if the eukaryote is a fungus, wherein the ⁇ -lactamase is a cytosolic ⁇ -lactamase; contacting a sample comprising the cell or an extract of the cell or conditioned medium with a substrate for ⁇ -lactamase of the invention; and determining the amount of substrate cleaved.
  • the amount of substrate cleaved is related to the amount of ⁇ -lactamase activity.
  • this invention provides methods for determining whether a test compound alters the expression of a gene operably linked to a set of expression control sequences.
  • the methods involve providing a cell transfected with a recombinant nucleic acid construct comprising the expression control sequences operably linked to nucleic acid sequences coding for the expression of a ⁇ -lactamase except if the eukaryote is a fungus, wherein the ⁇ -lactamase is a cytosolic ⁇ -lactamase; contacting the cell with the test compound; contacting a sample comprising the cell or an extract of the cell with a ⁇ -lactamase substrate of the invention; and determining the amount of substrate cleaved, whereby the amount of substrate cleaved is related to the amount of ⁇ -lactamase activity.
  • the step of determining the amount of substrate cleaved comprises exciting the compound; and determining the degree of fluorescence resonance energy transfer in the sample.
  • a degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of ⁇ -lactamase activity.
  • this invention provides methods of clonal selection comprising providing cells transfected with a recombinant nucleic acid molecule comprising the expression control sequences operably linked to nucleic acid sequences coding for the expression of a cytosolic ⁇ -lactamase; contacting the cells with a substance that activates or inhibits the activation of the expression control sequences; contacting the cells with a membrane-permeant compound of claim 1 which is converted into a substrate; determining whether substrate is cleaved within each individual cell, whereby cleavage reflects ⁇ -lactamase activity; selecting and propagating those cells with a selected level of ⁇ -lactamase activity.
  • the method further involves culturing selected cells in the absence of activator for a time sufficient for cleaved substrate to be substantially lost from the cells and for ⁇ -lactamse levels to return to unactivated levels; incubating the selected cells with a compound of claim 1 which is converted into a substrate; and selecting cells that have not substantially cleaved the substrate.
  • fluorescent donor moiety refers the radical of a fluorogenic compound which can absorb energy and is capable of transferring the energy to another fluorogenic molecule or part of a compound.
  • Suitable donor fluorogenic molecules include, but are not limited to, coumarins and related dyes, xanthene dyes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, and europium and terbium complexes and related compounds.
  • quencher refers to a chromophoric molecule or part of a compound which is capable of reducing the emission from a fluorescent donor when attached to the donor. Quenching may occur by any of several mechanisms including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling such as the formation of dark complexes.
  • acceptor refers to a quencher which operates via fluorescence resonance energy transfer. Many acceptors can re-emit the transferred energy as fluorescence.
  • Examples include coumarins and related fluorophores, xanthenes such as fluoresceins, rhodols, and rhodamines, resorufins, cyanines, difluoroboradiazaindacenes, and phthalocyanines.
  • Other chemical classes of acceptors generally do not re-emit the transferred energy. Examples include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, di- and triphenylmethanes.
  • die refers to a molecule or part of a compound which absorbs specific frequencies of light, including but not limited to ultraviolet light.
  • fluorophore refers to chromophore which fluoresces.
  • membrane-permeant derivative means a chemical derivative of a compound of general formula wherein at least one of X and Y contains at least one acylated aromatic hydroxyl, acylated amine, or alkylated aromatic hydroxyl wherein the acyl group contains 1 to 5 carbon atoms and wherein the alkyl group is selected from the group consisting of -CH 2 OC(O)alk, -CH 2 SC(O)alk, -CH 2 OC(O)Oalk, lower acyloxy-alpha-benzyl, and deltabutyrolactonyl; wherein alk is lower alkyl of 1 to 4 carbon atoms.
  • the masking groups are designed to be cleaved from the fluorogenic substrate within the cell to generate the derived substrate intracellularly. Because the substrate is more hydrophilic than the membrane permeant derivative it is now trapped within the cells.
  • alkyl refers to straight, branched, and cyclic aliphatic groups of 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 4 carbon atoms.
  • lower alkyl refers to straight and branched chain alkyl groups of 1 to 4 carbon atoms.
  • aliphatic refers to saturated and unsaturated alkyl groups of 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 4 carbon atoms.
  • ⁇ -Lactamases are nearly optimal enzymes in respect to their almost diffusion-controlled catalysis of ⁇ -lactam hydrolysis [Christensen, H. et al., Biochem. J. 266: 853-861 (1990)].
  • ⁇ -lactam hydrolysis Upon examination of the other properties of this class of enzymes, it was determined that they were suited to the task of an intracellular reporter enzyme. They cleave the ⁇ -lactam ring of ⁇ -lactam antibiotics, such as penicillins and cephalosporins, generating new charged moieties in the process [O'Callaghan, C.H. et al., Antimicrob.Agents.Chemother.
  • cephalosporin A first generation cephalosporin is illustrated below, left, with the arrow pointing to the site of cleavage by ⁇ -lactamase.
  • the free amino group thus generated donates electron density through the vinyl group to promote irreversible cleavage of a nucleofugal group R 2 from the 3'-position.
  • R 2 is thus free to diffuse away from the R 1 -cephalosporin conjugate (right-hand structure below).
  • ⁇ -Lactamases are a class of enzymes that have been very well characterized because of their clinical relevance in making bacteria resistant to ⁇ -lactam antibiotics [Waley, S.G., Sci. Prog. 72 : 579-597 (1988); Richmond, M.H. et al., Ann. N.Y. Acad. Sci. 182: 243-257 (1971)]. Most ⁇ -lactamases have been cloned and their amino acid sequence determined [see, e.g., Ambler, R.P., Phil. Trans. R. Soc. Lond. [Ser.B.] 289: 321-331 (1980)].
  • a gene encoding ⁇ -lactamase is known to molecular biologists as the ampicillin resistance gene (Amp r ) and is commonly used to select for successfully transduced bacteria [Castagnoli, L. et al., Genet.Res. 40 : 217-231 (1982)]; clones thereof are almost universally available.
  • the enzyme catalyzes the hydrolysis of a ⁇ -lactam ring and will not accept peptides or protein substrates [Pratt, R.F. and Govardhan, C.P., Proc. Natl. Acad. Sci.USA 81 : 1302-1306 (1984); Murphy, B.P.
  • the carboxyl group in the substrate can be easily masked by an acetoxymethyl ester [Jansen, A.B.A. and Russell, T.J., J.Chem.Soc. 2127-2132, (1965); Daehne, W. et al., J.Med.Chem. 13 : 607-612 (1970)], which is readily cleaved by endogenous mammalian intracellular esterases. Conversion by these esterases followed by the ⁇ -lactam cleavage by ⁇ -lactamase generates two negative charges and a tertiary amine, which protonates.
  • ⁇ -lactamases A large number of ⁇ -lactamases have been isolated and characterized, all of which would be suitable for use in accordance with the present invention.
  • ⁇ -lactamases were divided into different classes (I through V) on the basis of their substrate and inhibitor profiles and their molecular weight [Richmond, M.H. and Sykes, R.B., Adv.Microb.Physiol. 9 : 31-88 (1973)].
  • More recently, a classification system based on amino acid and nucleotide sequence has been introduced [Ambler, R.P., Phil. Trans. R. Soc. Lond. [Ser.B.] 289: 321-331 (1980)].
  • Class A ⁇ -lactamases possess a serine in the active site and have an approximate weight of 29kd.
  • This class contains the plasmid-mediated TEM ⁇ -lactamases such as the RTEM enzyme of pBR322.
  • Class B ⁇ -lactamases have an active-site zinc bound to a cysteine residue.
  • Class C enzymes have an active site serine and a molecular weight of approximately 39kd, but have no amino acid homology to the class A enzymes.
  • the coding region of an exemplary ⁇ -lactamase employed in the reporter gene assays described herein is indicated in SEQ ID NO:1 (nucleic acid sequence) and SEQ ID NO:2 (amino acid sequence).
  • SEQ ID NO:1 nucleic acid sequence
  • SEQ ID NO:2 amino acid sequence
  • the pTG2 del 1 containing this sequence has been described [Kadonaga, J.T. et al., J.Biol.Chem. 259: 2149-2154 (1984)].
  • the entire coding sequence of wildtype pBR322 ⁇ -lactamase has also been published [Sutcliffe, J.G., Proc.Natl.Acad.Sci.USA 75 : 3737-3741 (1978)].
  • ⁇ -lactamase reporter gene is employed in an assay system in a manner well known per se for the use of reporter genes (for example, in the form of a suitable plasmid vector).
  • ⁇ -lactamase substrates of the formula (I) exhibiting fluorescence resonance energy transfer (“FRET") when excited wherein:
  • linkers Z' and Z" serve the purpose of attaching the fluorescent donor and quencher moieties to the cephalosporin-derived backbone, and may facilitate the synthesis of the compounds of general formula I.
  • Z' may represent a direct bond to the backbone; alternatively, suitable linkers for use as Z' include, but are not limited to, the following: - (CH 2 ) n CONR 2 (CH 2 ) m -, -(CH 2 ) n NR 2 CO(CH 2 ) m -, -(CH 2 ) n NR 3 CONR 2 (CH 2 ) m -, -(CH 2 ) n NR 3 CSNR 2 (CH 2 ) m -, -(CH 2 ) n CONR 3 (CH 2 ) p CONR 2 (CH 2 ) m -, -(CH 2 ) n -, -(CH 2 ) n NR 3 CO(CH 2 ) p S(CH 2 ) p
  • Z" groups are-S(CH 2 ) n .
  • H is Especially preferred.
  • Preferred R' groups include H and methyl. Particularly preferred is H. Preferred R" groups include H and acetoxymethyl. A preferred R 2 group is H. A preferred A group is -S-.
  • the compounds of the present invention are membrane-permeant. Particularly preferred are such compounds wherein at least one of X and Y contains at least one acylated aromatic hydroxyl, acylated amine, or alkylated aromatic hydroxyl wherein the acyl group contains 1 to 5 carbon atoms and wherein the alkyl group is selected from the group consisting of -CH 2 OC(O)alk, -CH 2 SC(O)alk, -CH 2 OC(O)Oalk, lower acyloxy-alpha-benzyl, and deltabutyrolactonyl, wherein alk is lower alkyl of 1 to 4 carbon atoms.
  • Such compounds where at least one of X and Y contains at least one acylated aromatic hydroxy, wherein the acyl group is either acetyl, n -propionyl, or n -butyryl. Also particularly preferred are such compounds wherein at least one of X and Y contains an acetoxy methyl group on an aromatic hydroxyl group.
  • the acceptor is a fluorescein, rhodol, or rhodamin of formulae VIII-XII.
  • Preferred are such compounds where the donor is fluorescein of formula VIII and the acceptor is rhodol or rhodamine of formulae VIII-XII.
  • Also preferred are such compounds where the donor is a fluorescein of formula VIII and the acceptor is a tetrahalo fluorescein of formula VIII in which R a , R b , R c , and R d are independently Br or Cl.
  • the acceptor is a rhodol of formulae VIII, IX, and XI.
  • Another preferred group of such compounds are those where the acceptor is a rhodamine of formulae VIII, X, and XII.
  • the donor is a coumarin of formulae II-VII and the acceptor is a fluorescein, rhodol, or rhodamine of formulae VIII-XII, XLVII, or XLVII, and membrane-permeant fluorogenic derivatives thereof.
  • the acceptor is a fluorescein, rhodol, or rhodamine of formulae VIII-XII, XLVII, or XLVII, and membrane-permeant fluorogenic derivatives thereof.
  • fluorescein acceptor of formula VIII Particularly preferred are such compounds with a fluorescein acceptor of formula VIII.
  • the coumarin is 7-hydroxycoumarin or 7-hydroxy-6-chlorocoumarin and the fluorescein acceptor is fluorescein or dichlorofluorescein.
  • the efficiency of fluorescence resonance energy transfer depends on the fluorescence quantum yield of the donor fluorophore, the donor-acceptor distance and the overlap integral of donor fluorescence emission and acceptor absorption.
  • the energy transfer is most efficient when a donor fluorophore with high fluorescence quantum yield (preferably, one approaching 100%) is paired with an acceptor with a large extinction coefficient at wavelengths coinciding with the emission of the donor.
  • the dependence of fluorescence energy transfer on the above parameters has been reported [Forster, T.
  • R 0 The distance between donor fluorophore and acceptor dye at which fluorescence resonance energy transfer (FRET) occurs with 50% efficiency is termed R 0 and can be calculated from the spectral overlap integrals.
  • FRET fluorescence resonance energy transfer
  • the distance at which the energy transfer in this pair exceeds 90% is about 45 ⁇ .
  • the distances between donors and acceptors are in the range of 10 ⁇ to 20 ⁇ , depending on the linkers used and the size of the chromophores.
  • a chromophore pair will have to have a calculated R 0 of larger than 30 ⁇ for 90% of the donors to transfer their energy to the acceptor, resulting in better than 90% quenching of the donor fluorescence.
  • Cleavage of such a cephalosporin by ⁇ -lactamase relieves quenching and produces an increase in donor fluorescence efficiency in excess of tenfold. Accordingly, it is apparent that identification of appropriate donor-acceptor pairs for use as taught herein in accordance with the present invention would be essentially routine to one skilled in the art.
  • Chromophores suitable for use as X and Y are well known to those skilled in the art. Generic structures of particular classes of chromophores suitable for use as X and Y are provided below. Compounds of general formulas II - XXXIV are exemplary of fluorophores which serve as the basis for particularly suitable donor moieties in the compounds of general formula I. Suitable chromophores for use as the basis of acceptor moieties in the compounds of general formula include, but are not limited to, compounds of general formulas II - LIV. Chromophores of general formulae XXXV-LIV usually do not re-emit efficiently.
  • Europium tris-(bipyridine)cryptand donors may be suitably paired with acceptors of the formulae XV-XVII, XXXVI, XLVI-XLVII, LIV, and LVI.
  • Terbium tris-(bipyridine) cryptand donors may be suitably paired with acceptors of the formulae VIII-XVIII, XXXVI-XLI, and XLV-LIV, and LVI.
  • the Europium tris-(bipyridine) cryptand/ phtalocyanines donor/acceptor pair may be of particular interest when it is desirable to measure ⁇ -lactamase activity by emission of energy in the near to far red range.
  • any phenolic hydroxyls or free amines in the dye structures are acylated with C 1 -C 4 acyl groups (e.g. formyl, acetyl, n -butyl) or converted to various other esters and carbonates [for examples, as described in Bundgaard, H., Design of Prodrugs, Elsevier Science Publishers (1985), Chapter I, page 3 et seq.].
  • Phenols can also be alkylated with 1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, or methoxycarbonyloxymethyl groups.
  • fluoresceins, rhodols, and rhodamines this manipulation is particularly useful, as it also results in conversion of the acid moiety in these dyes to the spirolactone.
  • the carboxyl at the 4-position of the cephalosporin should be esterified with 1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl ,methylsulfinylmethyl, beta-morpholinoethyl, 2-(dimethylamino)ethyl, 2-(diethylamino)ethyl, or dialkylaminocarbonyloxymethyl groups as discussed in Ferres, H. (1980) Chem. Ind. 1980: 435-440.
  • the most preferred esterifying group for the carboxyl is acetoxymethyl.
  • RG is a nucleophile-reactive group (e.g., iodoacetamide, isocyanate, isothiocyanate, etc.); Nu is a nucleophile (e.g., -SH, -NH 2 , -OH, etc.); R 0 is H or an ester group (e.g., benzhydryl ester, tertiary butyl ester, etc.); Nu 0 is a bidentate nucleophile (e.g., HS - , HSCH 2 CH 2 NH 2 , xanthate, etc.); and Hal is a halogen (e.g., chlorine, bromine or iodine).
  • Nu is a nucleophile-reactive group (e.g., iodoacetamide, isocyanate, isothiocyanate, etc.); Nu is a nucleophile (e.g., -SH, -NH 2 , -OH, etc
  • cephalosporin starting materials are commercially available cephalosporin derivatives 7-aminocephalosporanic acid or 7-amino 3'-chlorocephalosporanic acid as its benzhydryl or tertiary butyl ester (R 0 ).
  • R 0 benzhydryl or tertiary butyl ester
  • dyes A and B carrying nucleophile reactive groups (RG) it is sometimes advantageous to esterify or alkylate their phenolic and free amine residues.
  • the order of attaching dye A and dye B depends on the choice of reagents.
  • Dye A is tethered to the cephalosporin via an alkyl amide linker.
  • dye A carrying a nucleophile-reactive group (RG) with a bifunctional aliphatic acid (e.g., amino-, mercapto- or hydroxyalkyl acid) and coupling of the acid to the cephalosporin 7-amine (path 1).
  • dye A carrying a nucleophilic group e.g., amine or thiol
  • path 2 the order of the two reactions can be reversed.
  • Dye A containing an aliphatic acid can be directly coupled to the cephalosporin (path 3).
  • Dye B carrying a nucleophilic substituent can be coupled to the 3'-position in the cephalosporin by direct displacement of the leaving group (LG) (path 4).
  • a Dye B carrying a nucleophile-reactive group can be reacted with a bidentate nucleophile which is coupled then attached to the cephalosporin by leaving group (LG) displacement (path 5); the order of the reactions can be reversed.
  • FRET fluorescent resonant energy transfer
  • Fluorescent donor moieties of particular interest include coumarins and fluoresceins.
  • Particular quenchers of interest include fluoresceins, rhodols and rhodamines.
  • Combinations of interest include the use of a coumarin donor with a fluorescein, rhodol or rhodamine quencher, and a fluorescein donor with a rhodol or rhodamine quencher.
  • a coumarin e.g., 7-hydroxycoumarin
  • chloro derivative thereof with a fluorescein or dichloro derivative thereof
  • fluorescein with an eosin or tetrachlorofluorescein a fluorescein with a rhodol derivative
  • fluorescein with a rhodamine with a fluorescein.
  • Europium chelate donors may be suitably paired with acceptors of the formulae XV-XVII, XXXVI, XLVI-XLVII, LIV, and LVI.
  • Terbium chelate donors may be suitably paired with acceptors of the formulae VIII-XVIII, XXXVI-XLI, and XLV-LIV, and LVI.
  • the europium and terbium chelate donors may be of particular interest for their very narrow emission peaks and their microsecond-to-millisecond excited state lifetimes, which can be readily discriminated from background fluorescence and scattering with excited-state lifetimes of nanoseconds or less.
  • any phenolic hydroxyls or free amines in the dye structures are acylated with C 1 -C 4 acyl groups (e.g. formyl, acetyl, n -butyryl) or converted to various other esters and carbonates for example, as described in Bundgaard, H., Design of Prodrugs, Elsevier Science Publishers (1985), Chapter 1, page 3 et seq.].
  • C 1 -C 4 acyl groups e.g. formyl, acetyl, n -butyryl
  • Phenols can also be alkylated with 1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, or methoxycarbonyloxymethyl groups.
  • acylation or alkylation of the free phenolic groups is particularly useful, as it also results in conversion of the acid moiety in these dyes to the spirolactone.
  • the carboxyl at the 4-position of the cephalosporin should be esterified with 1-(acyloxy)alkyl, acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulfinylmethyl, beta-morpholinoethyl, 2-(dimethylamino) ethyl, 2- (diethylamino) ethyl, or dialkylaminocarbonyloxymethyl groups as discussed in Ferres, H. (1980) Chem. Ind. 1980: 435-440.
  • the most preferred esterifying group for the carboxyl is acetoxymethyl.
  • the cephalosporin backbone serves as a cleavable linker between two dyes. After cleavage it provides the charges necessary to keep one of the two dyes inside the cell. Dyes are chosen in a manner that one dye absorbs light (acceptor chromophore) at the wavelength that the other one emits (donor fluorophore). In the intact cephalosporin the two dyes are in close proximity to each other. When exciting the donor fluorophore one observes fluorescence resonance energy transfer (FRET) from the donor to the acceptor instead of donor fluorescence [Forster, T., Ann. Physik 2 : 55-75 (1948)].
  • FRET fluorescence resonance energy transfer
  • acceptor is a nonfluorescent dye the energy is given off to the solvent; the donor fluorescence is quenched.
  • fluorescence re-emission occurs at the acceptor's emission wavelength.
  • hydrophobic donor and acceptor fluorophores can stack when separated by a short flexible linker. Due to this association in the ground state, a "dark complex" is formed [Yaron, A. et al., Anal. Biochem. 95: 228-235 (1979)]. In this complex, neither fluorophore can emit light, causing the fluorescence of both dyes to be quenched [Bojarski, C. and Sienicki, K.
  • Fluorescence resonance energy transfer has been used as a spectroscopic ruler for measuring molecular distances in proteins and peptides as it is effective in the range from 10-100 ⁇ . This energy transfer is proportional to the inverse sixth power of the distance between donor and acceptor. Its efficiency is higher, the better donor emission and acceptor absorbance overlap, and the longer the fluorescence lifetime of the donor (in absence of the acceptor). FRET can be very efficient over distances of 10-20 ⁇ .
  • distances for attachment of donor and acceptor are greater than 10 ⁇ and a minimum of 10 bond-lengths, if one includes the two minimal spacers at 7and 3-positions. Over this distance FRET is very efficient, if the right donor-acceptor pairs are chosen.
  • FRET-cephalosporin the 7-amine tethered dye stays attached to the polar hydrolysis products of cephalosporin cleavage, trapping it in the cells' cytoplasm. This position is best occupied by the donor fluorophore, although in some instances the acceptor may occupy this position. Upon cleavage, fluorescence increases due to loss of the quencher dye.
  • the acceptor fluorophore is generally attached by a linker which imparts the greatest stability of the substrate to nucleophilic attack.
  • a preferred linker is a thioether bond (-S-), which is very stable and due to its inductive effect reduces the reactivity of the ⁇ -lactam ring toward nucleophiles [Page, M.I., Adv.Phys.Org.Chem. 23 : 165-270 (1987)].
  • the free thiol or thiolate group released upon hydrolysis often quenches the attached fluorophore, adding to the desired large change in fluorescence upon hydrolysis.
  • the fluorogenic substrates of the invention are initially colorless and nonfluorescent outside cells.
  • the substrates are designed so they readily cross cell membranes into the cytoplasm, where they are converted to fluorescent compounds by endogenous nonspecific esterases and stay trapped due to their charges.
  • fluorescence energy transfer occurs leading to fluorescence at a particular wavelength when the substrates are excited. Lactamase cleavage of the ⁇ -lactam ring is followed by expulsion of the fluorescein moiety with loss of fluorescence energy transfer. Excitation of the modified substrate now results in fluorescence at a different wavelength.
  • the assay systems further provide an advantageous and rapid method of isolation and clonal selection of stably transfected cell lines containing reporter genes and having the desired properties which the transfection was intended to confer, e.g. fluorescent signal response after activation of a transfected receptor with a high signal-to-noise ratio from a high proportion of isolated cells.
  • Current procedures for clonal selection of satisfactorily transfected, genetically engineered cells from the initial population are done mainly by replica plating of colonies, testing of one set of colonies, visual selection of preferred clones, manual isolation of the replicas of the preferred clones by pipetting, and prolonged cellular cultivations.
  • the desired signal from cellular beta-lactamase reporter system can be maintained within living and viable cells. Replica plating of colonies is unnecessary because single cells can be assayed and remain viable for further multiplication.
  • a fluorescent-activated cell sorter e.g. the Becton Dickinson FACS VantageTM. The selected cells are then collected for cultivation and propagation to produce a clonal cell line with the desired properties for assays and drug screening.
  • the combination of a novel substrate in accordance with the invention and a suitable ⁇ -lactamase may be employed in a wide variety of different assay systems (such as are described in U.S. Patent 4,740,459).
  • the fluorogenic substrates of the invention enable the detection of ⁇ -lactamase activity in a wide variety of biologically important environments, such as human blood serum, the cytoplasm of cells and intracellular compartments; this facilitates the measurement of periplasmic or secreted ⁇ -lactamase.
  • any target protein can be detected by fusing a gene encoding the target protein to a ⁇ -lactamase gene, which can be localized by immunostaining and fluorescence or electron microscopy.
  • ⁇ -lactamase fusion proteins may be detected in the lumen of organelles through the use of the substrates of the invention; only subcellular compartments containing the fusion protein fluoresce at a wavelength characteristic of the cleaved substrate, whereas all others fluoresce at a wavelength characteristic of the intact molecule.
  • Both the intact and cleaved substrate are well retained in cells without the use of special measures, such as chilling.
  • the color change (even in individual small mammalian cells) is visible through a fluorescence microscope using normal color vision or photographic film; the fluorescence signal may be quantified and further enhanced by conventional digital image processing techniques.
  • gene activation is detected not by a change in a single intensity but rather by a color change or a change in the ratio between two intensities at different wavelengths, the assays of the present invention are relatively immune to many artifacts such as variable leakiness of cells, quantity of substrate, illumination intensity, absolute sensitivity of detection and bleaching of the dyes.
  • a variety of substrates have been prepared and their emission spectra obtained before and after ⁇ -lactamase cleavage. These substrates allow for ⁇ -lactamase detection primarily in vitro, as they bind strongly to serum and cellular proteins. Due to their hydrophobic nature, the fluorophores stack; this leads to a loss of fluorescence in the intact substrate. ⁇ -lactamase cleaves the substrates and relieves the stacking, allowing for fluorescence. Compounds (e.g., compound 11, Example 1) with reversed location of donor and acceptor fluorophore on the cephalosporin exhibit similar fluorescence behavior.
  • a compound of general formula 1 was coupled to a compound of general formula 2 to form a compound of general formula 3.
  • Commercially-available compound 4 was then coupled to compound 3 using dicyclohexylcarbodiimide and the product reacted with compound 5, yielding a compound of general formula 6.
  • Deprotection of compound 6 generated a compound of general formula 7.
  • Acyl was acetyl, R x was Me and R y H (a), or Acyl was butyryl, R x was H and R y Cl (b) ; R z was trimethylsilyl or benzyl.
  • the compounds of general formula 6 were modified to obtain membrane permeant derivatives which were converted to the corresponding fluorescent compounds of general formula 7 in intact cells due to the action of endogenous nonspecific esterases.
  • fluorescence resonance energy transfer occurs from the 7-hydroxycoumarin moiety to the fluorescein moiety, leading to green fluorescence when the compounds are excited at about 400 nm.
  • excitation of the 7-hydroxycoumarin moiety results in blue fluorescence; in exemplary embodiments, a 25-fold increase in fluorescence at about 450 nm and a three- to fourfold decrease in fluorescence at 515 nm was observed.
  • the substrates of this invention make it feasible to use ⁇ -lactamase as a reporter gene to monitor the expression from a set of expression control sequences.
  • this invention provides methods for monitoring gene expression from a set of expression control sequences by using ⁇ -lactamase as a reporter gene.
  • a cell is provided that has been transfected with a recombinant nucleic acid molecule comprising the expression control sequences operably linked to nucleic acid sequences coding for the expression of ⁇ -lactamase.
  • nucleic acid molecule includes both DNA and RNA molecules. It will be understood that when a nucleic acid molecule is said to have a DNA sequence, this also includes RNA molecules having the corresponding RNA sequence in which "U” replaces "T.”
  • recombinant nucleic acid molecule refers to a nucleic acid molecule which is not naturally occurring, and which comprises two nucleotide sequences which are not naturally joined together. Recombinant nucleic acid molecules are produced by artificial combination, e.g., genetic engineering techniques or chemical synthesis.
  • Nucleic acids encoding ⁇ -lactamases can be obtained by methods known in the art, for example, by polymerase chain reaction of cDNA using primers based on the DNA sequence in Fig. 1. PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).
  • Nucleic acids used to transfect cells with sequences coding for expression of the polypeptide of interest generally will be in the form of an expression vector including expression control sequences operatively linked to a nucleotide sequence coding for expression of the polypeptide.
  • nucleotide sequence "coding for expression of" a polypeptide refers to a sequence that, upon transcription and translation of mRNA, produces the polypeptide. As any person skilled in the are recognizes, this includes all degenerate nucleic acid sequences encoding the same amino acid sequence. This can include sequences containing, e.g., introns.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which they are operatively linked. Expression control sequences are "operatively linked" to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.
  • the recombinant nucleic acid can be incorporated into an expression vector comprising expression control sequences operatively linked to the recombinant nucleic acid.
  • the expression vector can be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, markers, etc.
  • the recombinant nucleic acid used to transfect the cell contains expression control sequences operably linked to a nucleotide sequence encoding a ⁇ -lactamase.
  • the ⁇ -lactamase encoded can be any known to the art or described herein. This includes, for example, the enzymes shown in Fig. 7.
  • cytosolic ⁇ -lactamase refers to a ⁇ -lactamase that lacks amino acid sequences for secretion from the cell membrane, e.g., the signal sequence.
  • the signal sequence For example, in the polypeptide of Sequence 1 of Fig. 7, the signal sequence has been replaced with the amino acids Met-Ser. Accordingly, upon expression, this ⁇ -lactamase remains within the cell.
  • nucleic acid molecules including expression control sequences adapted for function in a mammalian eukaryotic cell operably linked to a nucleotide sequence coding for the expression of a ⁇ -lactamase can be used in the method of the present invention.
  • the ribosome binding site and nucleotide sequence coding for expression of ⁇ -lactamase contain sequences preferred by mammalian cells. Such sequences improve expression of ⁇ -lactamase in mammalian cells. Preferred sequences for expression in mammalian cells are described in, for example, Kozak, M., J. Cell Biol. 108: 229-241 (1989), referred to herein as "Kozak sequences".
  • the nucleotide sequence for cytosolic ⁇ -lactamase in Sequence 3 of Fig. 7 contains Kozak sequences for the nucleotides -9 to 4 (GGTACCACCATGA).
  • the expression control sequences When used in mammalian cells, the expression control sequences are adapted for function in mammalian cells.
  • the method of this invention is useful to testing expression from any desired set of expression control sequences.
  • this invention is useful for testing expression from inducible expression control sequences.
  • inducible expression control sequences refers to expression control sequences which respond to biochemical signals either by increasing or decreasing the expression of sequences to which they are operably linked.
  • the expression control sequences include hormone response elements. The binding of a steroid hormone receptor to the response element induces transcription of the gene operably linked to these expression control sequences.
  • Expression control sequences for many genes and for inducible genes, in particular, have been isolated and are well known in the art.
  • the invention also is useful with constitutively active expression control sequences.
  • the transfected cell is incubated under conditions to be tested for expression of ⁇ -lactamase from the expression control sequences.
  • the cell or an extract of the cell is contacted with a ⁇ -lactamase substrate of the invention under selected test conditions and for a period of time to allow catalysis of the substrate by any ⁇ -lactamase expressed.
  • the donor moiety from this sample is excited with appropriate ultraviolet or visible wavelengths. The degree of fluorescence resonance energy transfer in the sample is measured.
  • this method can be used to compare mutant cells to identify which ones possess greater or less enzymatic activity. Such cells can be sorted by a fluorescent cell sorter based on fluorescence.
  • reporter gene cell-based assays for screening samples or pools of samples (such as compounds (combinatorial or synthetic), natural product extracts, or marine animal extracts) to identify potential drug candidates which act as agonists, inverse agonists or antagonists of cellular signaling or activation
  • the combination of cells (preferably mammalian) genetically engineered to express beta-lactamase under the control of different regulatory elements/promoters and the use of the novel beta-lactamase substrate compounds of the present invention will provide distinct advantages over known reporter genes (including, but not limited to, chloramphenicol acetyl transferase, firefly luciferase, bacterial luciferase, Vargula luciferase, aequorin, beta-galactosidase, alkaline phosphatase) and their requisite substrates.
  • assays can be constructed to detect or measure the ability of test substances to evoke or inhibit functional responses of intracellular hormone receptors.
  • these include expression control sequences responsive to induction by mineralcorticosteroids, including dexamethasone [J. Steroid Biochem. Molec. Biol. Vol. 49, No. 1 1994, pp.31-3]), gluococorticoid, and thyroid hormone receptors [as described in US patent 5,071,773].
  • Additional such intracellular receptors include retinoids, vitamin D3 and vitamin A [Leukemia vol 8, Suppl. 3, 1994 ppS1-S10; Nature Vol.
  • promoters such as c-fos or c-jun [US patent 5,436,128; Proc. Natl. Acad. Sci. Vol. 88, 1991, pp. 5665-5669] or promoter constructs containing regulatory elements responsive to second messengers [ Oncogene, 6 : 745-751 (1991)] (including cyclic AMP-responsive elements, phorbol ester response element (responsive to protein kinase C activation), serum response element (responsive to protein kinase C-dependent and independent pathways) and Nuclear Factor of Activated T-cells response element (responsive to calcium)) to control expression of beta-lactamase
  • assays can be constructed to detect or measure substances or mixtures of substances that modulate cell-surface receptors including, but not limited to, the following classes: receptors of the cytokine superfamily such as erthyropoietin, growth hormone, interferons, and interleukins (other than IL-8) and colony-stimulating
  • assays can be constructed to identify substances that modulate the activity of voltage-gated or ligand-gated ion channels, modulation of which alters the cellular concentration of second messengers, particularly calcium [US patent 5,436,128]. Assays can be constructed using cells that intrinsically express the promoter, receptor or ion channel of interest or into which the appropriate protein has been genetically engineered.
  • the expression control sequences also can be those responsive to substances that modulate cell-surface receptors or that modulate intra-cellular receptors.
  • cells containing beta-lactamase controlled by a desired promoter/enhancer element are incubated with test substance(s), substrate then added, and after a certain period of time the fluorescence signal is measured at either one or two excitation-emission pairs appropriate to the chosen compound of the invention (e.g. compound CCF2 with wavelength pairs of near 405 nm and near 450 nm and near 405 and near 510 nm).
  • This fluorescent result is compared to control samples which have had no drug treatment and, when feasible, control samples with a known inhibitor and a known activator.
  • any active drugs is then determined using the ratio of the fluorescence signal found in test wells to the signals found in wells with no drug treatment.
  • Assays are performed in wells in a microtiter plate containing 96 or more wells or in an assay system with no compartments such as a gel matrix or moist membrane environment. Detection could be done for example by microtiter plate fluorimeters, e.g. Millipore Cytofluor, or imaging devices capable of analyzing one or more wells or one or more assay points in a certain surface area, e.g. as supplied by Astromed.
  • the ability to retain the substrate in the cytoplasm of living cells is advantageous as it can allow a reduction in signal interference from coloured or quenching substances in the assay medium.
  • the fluorescent signal from the compounds of this invention can be readily detected in single cells and thus allowing assay miniaturization and an increased number of tests per surface area.
  • Miniaturized assays also further increase the throughput of an imaging detection system as there are more samples within the imaging field.
  • the assay systems further provide an advantageous and rapid method of isolation and clonal selection of stably transfected cell lines containing reporter genes and having the desired properties which the transfection was intended to confer, e.g. fluorescent signal response after activation of a transfected receptor with a high signal-to-noise ratio of at least 10:1 from a high proportion of isolated cells.
  • Current procedures for clonal selection of satisfactorily transfected, genetically engineered cells from the population initial transfected with the vectors of interest are done mainly by manual means and involve several rounds of microscopic analyses, selecting the visually preferred clone, isolation of the clone by manual pipetting stages and prolonged cellular cultivations.
  • the desired signal from cellular beta-lactamase reporter system can be maintained within living and viable cells.
  • a fluorescent-activated cell sorter e.g. the Becton Dickinson FACS Vantage.
  • the selected cells are then collected for cultivation and propagation to produce a clonal cell line with the desired properties for assays and drug screening.
  • the presence (for example, in human serum, pus or urine) of bacteria resistant to ⁇ -lactam antibiotics can be readily detected using the substrates of the present invention. Only in the presence of an active ⁇ -lactamase is there a change in the fluorescence spectrum from that of the intact molecule to one characteristic of the cleavage product.
  • the substrates of the present invention are superior to prior art chromogenic substrates Nitrocephin and PADAC, in that the inventive substrates are stable to human serum.
  • the novel substrates are also more sensitive than the chromogenic substrate CENTA, because they experience a much smaller optical background signal from human serum and a lower detection limit for fluorescence versus absorbance.
  • the degree of FRET can be determined by any spectral or fluorescence lifetime characteristic of the excited construct, for example, by determining the intensity of the fluorescent signal from the donor, the intensity of fluorescent signal from the acceptor, the ratio of the fluorescence amplitudes near the acceptor's emission maxima to the fluorescence amplitudes near the donor's emission maximum, or the excited state lifetime of the donor. For example, cleavage of the linker increases the intensity of fluorescence from the donor, decreases the intensity of fluorescence from the acceptor, decreases the ratio of fluorescence amplitudes from the acceptor to that from the donor, and increases the excited state lifetime of the donor.
  • changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing.”
  • ratioing Changes in the absolute amount of substrate, excitation intensity, and turbidity or other background absorbances in the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately in parallel. Therefore the ratio of the two emission intensities is a more robust and preferred measure of cleavage than either intensity alone.
  • the excitation state lifetime of the donor moiety is, likewise, independent of the absolute amount of substrate, excitation intensity, or turbidity or other background absorbances. Its measurement requires equipment with nanosecond time resolution, except in the special case of lanthanide complexes in which case microsecond to millisecond resolution is sufficient.
  • Fluorescence in a sample is measured using a fluorimeter.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics cause the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • silica gel chromatography was performed using silica gel (Merck, grade 60, 230-400 mesh, 60 ⁇ ) purchased from Aldrich. Bakerbond Octadecyl from J. T. Baker was used for C 18 reverse phase chromatography. Solvents (high pressure liquid chromatography grade) were used for chromatography as received, or dried over activated molecular sieves (3 ⁇ ) for synthetic purposes.
  • Fluorescence excitation and emission spectra were measured either on a Spex Fluorolog 111 or on a K2 fluorometer (ISS, Champaigne, IL) in ratio mode with a rhodamine B quantum counter.
  • the efficiency of fluorescence energy transfer was determined from the change in the integrated fluorescence emission at the donor emission wavelength upon treatment with ⁇ -lactamase.
  • fluorescence microscopy imaging two different imaging setups were used. One, with an inverted fluorescence microscope, Zeiss IM-35 (Thornwood, NY) coupled to a silicon-intensified target (SIT) camera (Dage-MTI, Michigan City, IN) has been described in detail [Tsien, R.Y.
  • Fluorescence resonance energy transfer was measured by monitoring the ratio of fluorescence intensities at donor and acceptor emission wavelengths using commercially-available filters (Omega Optical).
  • the first synthesis step was to convert 7-aminocephalosporanic acid into a bifunctional cephalosporin carrying a thiol in the 3'-position and the 7-amine [Van Heyningen, E. and Brown, C.N., J.Med.Chem. 8 : 174-181 (1965); Japanese Patent, Kokai 75/18494, CA 85 , 97320d].
  • This cephalosporin was then reacted selectively with an thiol-reactive dye, followed by an amine-reactive dye.
  • the thiol-reactive dye 5,(6)-iodoacetamido-fluorescein and the amine-reactive dye 5,(6)-carboxy-N,N,N',N'-tetramethylrhodamine-succinimide were coupled to the cephalosporin in aqueous dimethylformamide at pH 8.
  • the product will be referred to as RCF.
  • the thiomethyl linker was introduced by conversion of 5-fluoresceinamine to 5-mercaptofluorescein via diazotization, conversion to the ethylxanthate, and degradation of the xanthate by aqueous acid to the free sulfhydryl. It was coupled to 7-bromoacetamidocephalosporanic acid by nucleophilic displacement of the bromide by the mercapto group of the fluorescein. 7-Bromoacetamido-cephalosporanic acid had been prepared from 7-aminocephalosporanic acid and bromoacetyl bromide [Bunnell, C.A. et al. Industrial manufacture of cephalosporins.
  • Beta-Lactam Antibiotics for Clinical Use Series: Clinical Pharmacology Vol. 4, edited by Queener, S.F., Webber, J.A. and Queener, S.W. New York: M. Dekker, 1986, p. 255-283].
  • 5-Fluoresceinamine was brominated to generate 5-eosinamine, which was converted into 5-mercaptoeosin in analogous way to the 5-mercaptofluorescein.
  • the FRET-cephalosporin was generated as the protected tetraacetyl derivative.
  • 5-eosinamine To prepare 5-eosinamine, 1.74g (5mmol) 5-fluoresceinamine was suspended in 30ml glacial acetic and 2.06ml (40mmol, 100% excess) bromine was added. With the addition of bromine the fluoresceinamine went into solution. The solution was heated for six hours at 90°C, during which period a white precipitate began to form. An ice-cooled trap attached to the flask kept bromine from escaping into the atmosphere. Excess bromine was then recovered by distillation into a liquid nitrogen cooled collecting flask. One volume of water was added to the acetic acid solution to precipitate any product remaining in solution. The precipitate was collected by filtration and dissolved in 1N aqueous sodium hydroxide.
  • 5-Eosinamine was precipitated as the free amine by addition of glacial acetic acid.
  • the eosinamine was dissolved in little chloroform and methanol was added. Concentrating this solution on the rotary evaporator gave 2.56g (3.85mmol, 77%) eosinamine as a fine white powder (the eosinaminespirolactone).
  • 5-eosin-ethylxanthate diacetate 670mg (1mmol) 5-eosinamine were stirred in 2ml concentrated sulfuric acid and 2ml glacial acetic acid. The suspension was cooled with an ice-salt bath to a few degrees below 0°C, which turned it into a thick paste that was difficult to stir. 200mg (2.9mmol) sodium nitrite in 1ml water were added dropwise over the period of one hour. After another 2 hours at 0°C 20g of ice was slowly added. The flask was put on the high vacuum pump in the cold, to remove excess nitrous gases (caution!).
  • the residue was flash-chromatographed over silica gel with ethyl acetate-methanol-acetic acid (100:1:1) as the eluent.
  • Deprotection of the acetates was achieved by incubating the product with orange peel acetylesterase in 50mM phosphate buffer (pH7) for 24 hours at 37°C.
  • the deacylated product was purified by C 18 reverse phase chromatography.
  • the eluent was a step gradient of 25mM aqueous phosphate buffer (pH7) and methanol. Fluorescein byproducts eluted with 33% and 50% methanol in the eluent, after which the desired product eluted in 66% methanol.
  • the deprotected compound shows little fluorescence in phosphate buffer as the two hydrophobic dyes stack. The remaining fluorescence is due to fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • Cleavage of the compound increases fluorescence at 515nm about 70-fold (Fig. 2).
  • the fluorescence properties of the compound can be attributed to dye-dimer formation, as FRET increases drastically once methanol is added to the solution. Methanol breaks the hydrophobic interaction that causes the fluorophores to stack.
  • the cephalosporin 7-amine was reacted with bromoacetyl bromide in aqueous dioxane, followed by bromide displacement with 5-fluoresceinthiol to yield a FRET-cephalosporin that is virtually nonfluorescent in 50mM phosphate buffer pH 7. This compound is referred to as FCRX.
  • the first step in preparation of 5-rhodol-X-bromoacetamide was synthesis of 9-(2'-carboxy-4'(5')-nitro-benzoyl)-8-hydroxyjulolidine and separation of the isomers.
  • 10.1g 48mmol,92% purity
  • 4-Nitrophthalic anhydride were dissolved in 20ml toluene at 70°C.
  • 9.76g 50mmol, 97% purity
  • 8-Hydroxyjulolidine in 20ml ethyl acetate were added and the solution kept at 70°C for 30min.
  • the reaction mixture was run through a short bed of silica gel followed by ethyl acetate as eluent.
  • the above product (19) was dissolved in 1ml dioxane-water (1:1) with 20mg sodium bicarbonate. 10 ⁇ l Bromoacetyl bromide were added to the solution on ice. The solution was kept for another 1.5 hours at room temperature. 20mg sodium bicarbonate and 10 ⁇ l bromoacetyl bromide were added to the solution with ice cooling. After another 1.5 hours at room temperature the dioxane was removed on the rotary evaporator and the products were precipitated from the aqueous solution with 1M phosphoric acid and collected by centrifugation. The solids were suspended in dilute aqueous bicarbonate solution and the undissolved particles removed by centrifugation and discarded. The product was precipitated with 1M phosphoric acid and purified by flash chromatography on silica gel with chloroform-methanol-acetic acid-water (55:15:4:2). This procedure dissolved small amounts of silica gel.
  • Fig. 3 shows the fluorescence emission spectra of this FRET-cephalosporin in 50mM phosphate buffer pH 7 before and after treatment with ⁇ -lactamase.
  • the low initial fluorescence is due to the stacking of the fluorophores, forming a ground state complex that is nonfluorescent. When one adds methanol to the solution this stacking can be disrupted and efficient fluorescence resonance energy transfer occurs.
  • FCRE N- [resorufin-4-carbonyl] -N'-iodoacetyl-piperazine
  • the FRET-cephalosporin FCRE (25) carrying fluorescein as the donor and resorufin as the quencher was made by the same procedure as the one carrying the rhodol-X-acceptor.
  • the N-[resorufin-4-carbonyl]-N'-iodoacetylpiperazine (Boehringer Mannheim) was coupled to the free 3'-thiol of the cephalosporin followed by bromoacetylation and addition of the 5-fluoresceinthiol.
  • three equivalents of 5-fluorescein thiol were added, as the first equivalent instantaneously reduced the resorufin and formed unreactive difluorescein-disulfide.
  • a toluene solution of O-benzylglycine [prepared by extraction of 3.4 g (10 mMol) benzylglycine tosyl salt with ethyl acetate - toluene - saturated aqueous bicarbonate - water (1 : 1 : 1 : 1, 250 ml), drying of the organic phase with anhydrous sodium sulfate and reduction of the solvent volume to 5 ml] was added dropwise to the coumarin solution. The reaction was kept at room temperature for 20 hours after which the precipitate was removed by filtration and washed extensively with ethylacetate and acetone.
  • 7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin was prepared as follows. 920 mg (2 mMol) 7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin were dissolved in 50 ml dioxane. 100 mg palladium on carbon (10%) and 100 ⁇ l acetic acid were added to the solution and the suspension stirred vigorously in a hydrogen atmosphere at ambient pressure. After the uptake of hydrogen ceased the suspension was filtered. The product containing carbon was extracted five times with 25 ml boiling dioxane. The combined dioxane solutions were let to cool upon which the product precipitated as a white powder.
  • Deprotection of the fluorescein acetates in compound 27 was accomplished with sodium bicarbonate in methanol (room temperature, 30minutes) to provide the fluorescent enzyme substrate CCF2. It was purified by high performance liquid chromatography on a reverse phase C 18 - column using 35% aqueous acetonitrile containing 0.5% acetic acid as the eluent.
  • the donor and acceptor dyes in substrate CCF2 do not stack.
  • the substrate is fully fluorescent in phosphate buffer and there is no formation of the "dark complex" (i.e., addition of methanol does not change the fluorescence spectrum of CCF2, except for the effect of dilution). This is due to the much smaller and more polar nature of the 7-hydroxycoumarin compared to that of the xanthene dyes (eosin, rhodamine, rhodol and resorufin) in Examples 1-4.
  • Fig. 5 illustrates the emission spectrum of compound CCF2 in 50 mmolar phosphate buffer pH 7.0 before and after ⁇ -lactamase cleavage of the ⁇ -lactam ring.
  • efficient energy transfer occurs from the 7-hydroxycoumarin moiety to the fluorescein moiety.
  • Excitation of the substrate at 405 nm results in fluorescence emission at 515 nm (green) from the acceptor dye fluorescein.
  • the energy transfer is disrupted when ⁇ -lactamase cleaves the ⁇ -lactam ring, thereby severing the link between the two dyes.
  • Excitation of the products at 405 nm now results entirely in donor fluorescence emission at 448 nm (blue).
  • the fluorescence emission from the donor moiety increases 25 fold upon ⁇ -lactam cleavage.
  • the fluorescence at 515 nm is reduced by 3.5 fold, all of the remaining fluorescence originating from the 7-hydroxycoumarin as its emission spectrum extends into the green.
  • Twenty-five-fold quenching of the donor in the substrate is equivalent to an efficiency of fluorescence energy transfer of 96%. This large fluorescence change upon ⁇ -lactam cleavage can readily be used to detect ⁇ -lactamase in the cytoplasm of living mammalian cells, as is reported in Examples 6 and 7.
  • the 7-hydroxycoumarin moiety in the cephalosporin was determined to have a fluorescence quantum efficiency in the absence of the acceptor of 98-100%. This value was determined by comparing the integral of the corrected fluorescence emission spectrum of the dye with that of a solution of 9-aminoacridine hydrochloride in water matched for absorbance at the excitation wavelength. It follows that 7-hydroxycoumarin is an ideal donor dye, as virtually every photon absorbed by the dye undergoes fluorescence energy transfer to the acceptor.
  • Cells of the T-cell lymphoma line Jurkat were suspended in an isotonic saline solution (Hank's balanced salt solution) containing approximately 10 12 ⁇ -lactamase enzyme molecules per milliliter (approximately 1.7 nM; Penicillinase 205 TEM R + , from Sigma) and 1 mg/ml rhodamine conjugated to dextran (40 kd) as a marker of loading.
  • the suspension was passed through a syringe needle (30 gauge) four times. This causes transient, survivable disruptions of the cells' plasma membrane and allows entry of labeled dextran and ⁇ -lactamase.
  • Cells that had been successfully permeabilized contained ⁇ -lactamase and were red fluorescent when illuminated at the rhodamine excitation wavelength on a fluorescent microscope.
  • the cells were incubated with 5 ⁇ M fluorogenic ⁇ -lactamase substrate, CCF2/ac 2 AM 2 , at room temperature for 30 minutes. Illumination with violet light (405 nm) revealed blue fluorescent and green fluorescent cells. All cells that had taken up the marker rhodamine-dextran appeared fluorescent blue, while cells devoid the enzyme appeared fluorescent green.
  • Cells from cell lines of various mammalian origin were transiently transfected with a plasmid containing the RTEM ⁇ -lactamase gene under the control of a mammalian promotor.
  • the gene encodes cytosolic ⁇ -lactamase lacking any signal sequence and is listed as SEQ. ID. 1. 10 to 48 hours after transfection cells were exposed to 5 ⁇ mol CCF2/ac 2 AM 2 for 1 to 6 hours. In all cases fluorescent blue cells were detected on examination with a fluorescence microscope. Not a single blue fluorescent cell was ever detected in nontransfected control cells.
  • COS-7 (origin: SV40 transformed african green monkey kidney cells) Table of pixel intensities coumarin emission filter fluorescein emission filter Blue cell #1 27 20 #2 34 23 #3 31 31 #4 22 33 Green cell#1 4 43 #2 4 42 #3 5 20 #4 3 24 CHO (origin: Chinese hamster ovary cells) Table of pixel intensities coumarin emission filter fluorescein emission filter Blue cell #1 98 112 #2 70 113 #3 76 92 #4 56 67 Green cell #1 9 180 #2 9 102 #3 7 101 #4 9 83
  • the solid was dissolved in 30 ml ethylacetate-acetonitrile (2:1) and the solution extracted twice with an equal volume of 1 N hydrochloric acid and then with brine. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the solid crystallized from boiling ethylacetate with addition of hexane. The yield was 316mg (1.0 mMol, 63%) of a white crystalline solid.
  • Cleavage of the diphenylmethyl ester to give CCFlac 3 was effected as follows. 4 mg (4 ⁇ Mol) of CCFlac 3 diphenylmethyl ester were treated with 200 ⁇ l trifluoroacetic acid - anisole - methylene chloride (10:1:10) on ice for 15 minutes. The reagents were removed in vacuo and the residue was dissolved in 0.5 ml ethyl acetate and the solvent evaporated in vacuo. The solid was triturated with ether and then dissolved in 0.5 ml methanol. Addition of the methanolic solution to 2 ml water precipitated the product. The product was recovered by centrifugation and dried in vacuo.
  • the yield was 2mg (2 ⁇ Mol, 50%) white solid.
  • the compound was further purified by high performance liquid chromatography on a reverse phase C 18 -column using 55% aqueous acetonitrile containing 0.5% acetic acid as the eluent.
  • the fluorescence emission spectrum of CCF1 before and after ⁇ -lactamase cleavage was obtained from a sample of CCF1ac 3 that had been converted to CCF1 by treatment with orange peel acetyl esterase in 50 mmolar aqueous phosphate buffer pH 7.
  • Substrate CCF1 has similar fluorescence properties to substrate CCF2 in Example 5.
  • efficient energy transfer occurs from the 7-hydroxycoumarin moiety to the fluorescein moiety.
  • Excitation of the substrate at 390 nm results in fluorescence emission at 515 nm (green) from the acceptor dye fluorescein.
  • the energy transfer is disrupted when ⁇ -lactamase cleaves the ⁇ -lactam ring, thereby severing the link between the two dyes.
  • Excitation of the products at 390 nm now results entirely in donor fluorescence emission at 460 nm (blue).
  • the fluorescence emission from the donor moiety increases 25-fold upon ⁇ -lactam cleavage.
  • the fluorescence at 515 nm is reduced by 3-fold, all of the remaining fluorescence originating from the 7-hydroxycoumarin as its emission spectrum extends into the green. Twenty-five-fold quenching of the donor in the substrate is equivalent to an efficiency of fluorescence energy transfer of 96%. This large fluorescence change upon ⁇ -lactam cleavage can readily be used to detect ⁇ -lactamase in the cytoplasm of living mammalian cells, as is reported in Example 9.
  • Cells of the T-cell lymphoma line Jurkat were suspended in an isotonic saline solution (Hank's balanced salt solution) containing approximately 10 12 ⁇ -lactamase enzyme molecules per milliliter (approximately 1.7 nM; Penicillinase 205 TEM R + , from Sigma) and 1 mg/ml rhodamine conjugated to dextran (40 kd) as a marker of loading.
  • the suspension was passed through a syringe needle (30 gauge) four times. This causes transient, survivable disruptions of the cells' plasma membrane and allows entry of labeled dextran and ⁇ -lactamase.
  • Cells which had been successfully permeabilized contained ⁇ -lactamase and were red fluorescent when illuminated at the rhodamine excitation wavelength on a fluorescent microscope.
  • the cells were incubated with 30 ⁇ M fluorogenic ⁇ -lactamase substrate CCFlac 3 at room temperature for 30 minutes. Illumination with ultraviolet light (360 nm) revealed blue fluorescent and green fluorescent cells. All cells that had taken up the marker rhodamine-dextran appeared fluorescent blue, while cells devoid the enzyme appeared fluorescent green.
  • the preferred membrane-permeable ester of CCF2 was prepared as follows:
  • Activation of the intracellular glucocorticoid receptor was measured by its ability to upregulate the transcriptional activity of the glucocorticoid responsive element in the mouse mammary tumor virus promotor. This response to steroids was detected as increased intracellular ⁇ -lactamase activity on the substrate CCF2 causing an appropriate change in fluorescent signal.
  • the gene for plasmid encoded RTEM ⁇ -lactamase of Escherichia coli without a signal sequence was put under transcriptional control of the mouse mammary tumor virus promotor and introduced into a mammalian expression vector.
  • This vector also carried the chloramphenicol resistance marker for amplification of the plasmid in bacteria and the neomycin resistance marker for mammalian selection. It was introduced into baby hamster kidney (BHK) cells in culture using the calcium phosphate precipitation technique. Cells were then subjected to selection for stable integration of the plasmid into the cells' genome using the antibiotic G418.
  • One of twenty clones was selected for its marked increase in ⁇ -lactamase expression following exposure to the steroid analog dexamethasone.
  • Activation of cell surface receptors leads to a change in intracellular messenger concentrations which in turn modulates intracellular transcription factor activity.
  • an increase the intracellular concentration of the messenger ion calcium leads to the activation of the nuclear factor of activated T-lymphocytes (NFAT). This event increases transcription at promoters containing the NFAT-recognition site.
  • NFAT nuclear factor of activated T-lymphocytes
  • An increase in calcium levels alone is sufficient to markedly increase transcription of a reporter gene such as ⁇ -lactamase regulated when it is put under transcriptional control of a promotor containing a trimer of NFAT sites.
  • the murine T-lymphocyte cell line B3Z was transiently cotransfected with two plasmids.
  • One plasmid contained the ⁇ -adrenergic receptor, which localizes at the cells' surface, under the transcriptional control of the strong and constitutively active cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the other plasmid contained the bacterial RTEM ⁇ -lactamase gene from Escherichia coli modified for improved mammalian expression (sequence ID # 3 , with optimum mammalian Kozak sequence, ⁇ -globin leader sequence, pre-sequence removed) under the transcriptional control of a promotor containing a trimer of NFAT sites.
  • the plasmids were introduced into cells using electroporation. 5x10 6 cells in 0.5 ml electroporation buffer were electroporated in the presence of 10 ⁇ g each of both plasmids using the Biorad Gene Pulser (250V, 960 ⁇ F, 16 ⁇ sec). Twenty-four hours after transfection, cells were either incubated in the presence or absence of the ⁇ -adrenergic agonist isoproterenol (10 ⁇ molar) for 5 hours. The supernatant was removed and replaced with Hank's balanced salt solution containing 10 ⁇ molar CCF2/btAMac 2 . After 20 minutes at room temperature cells were washed with fresh buffer and viewed with the fluorescence microscope.
  • ⁇ -Lactamases from different microorganisms were modified for use as reporter enzymes in eukaryotic cells, preferably mammalian.
  • the bacterial gene for these enzymes includes a N-terminal pre-sequence (first 23 amino acids of Sequence 2 of Fig. 7.) that targets the enzyme to the extracellular space. Following translocation a pre-sequence peptidase cleaves the 23 amino acid pre-sequence releasing the mature ⁇ -lactamase enzyme.
  • RTEM ⁇ -lactamase from Escherichia coli including its bacterial pre-sequence (Sequence 2 of Fig. 7) was put into a mammalian expression vector under the control of the mouse mammary tumor virus promotor.
  • This construct was introduced into baby hamster kidney cells using the standard calcium phosphate precipitation technique.
  • the ⁇ -lactamase activity was found in the cell culture medium; no activity could be detected in the cell pellet.
  • the amount of ⁇ -lactamase activity in the medium was steroid dependent. Cells that had been in the presence of 1 ⁇ M dexamethasone for 36 hours prior to the measurement produced threefold more enzyme than control cells. This makes the ⁇ -lactamase with its bacterial pre-sequence (Sequence 2 of Fig. 7) useful for an extracellular assay of mammalian reporter gene activity.
  • a preferred use of the ⁇ -lactamase reporter is where the enzyme is produced and retained in the cell cytoplasm. Therefore the bacterial signal sequence was removed and replaced by ATG (methionine) as the new translational start site in three modified RTEM ⁇ -lactamase genes (Sequences 1, 3, and 4 of Fig. 7).
  • ATG methionine
  • the RTEM ⁇ -lactamases of Sequences 3 and 4 of Fig. 7 were constructed with altered ribosome binding sites optimized for mammalian expression [Kozak, M., J . Cell Biol. 108: 229-241 (1989)].
  • ⁇ -lactamase of sequence ID #3 was inserted at the end of an untranslated mammalian ⁇ -globin leader sequence. All of these novel DNA sequences encoding novel ⁇ -lactamases were inserted into mammalian expression vectors with the cytomegalovirus promotor controlling their transcription.
  • Mammalian cells in tissue culture Hela, COS-7, CHO, BHK were transfected transiently with the plasmids using the standard lipofectin technique. Two to five days after transfection, the cells were incubated with the membrane-permeant derivative, CCF2/btAMac 2 , of the fluorescent substrate CCF2 to assay functional expression of the enzyme.
  • the gene for Bacillus licheniformis ⁇ -lactamase was isolated from total Bacillus licheniformis DNA by use of the polymerase chain reaction.
  • the oligonucleotide primers removed the ⁇ -lactamase secretion sequence and generated the DNA sequence ID # 5.
  • This gene was inserted in a pCDNA3 mammalian expression vector under the transcriptional control of the constitutively active cytomegalovirus promoter.
  • HeLa cells were transfected with 10 ⁇ g of plasmid per 25 cm 2 culture dish using lipofectin.
  • a plasmid was constructed with ⁇ -lactamase of sequence ID 3 (figure 7) under control of yeast elongation factor EF-lalpha enhancer and promoter. This plasmid was coinjected together with the potassium salt of substrate CCF2 (compound 7b) into zebrafish embryos at the single cell stage. As control, embryos were injected with the potassium salt of substrate CCF2 alone. After three hours, the embryos were viewed with an epifluorescence microscope using violet excitation light (filter 400DF15) and a 435 nm longpass emission filter. Embryos that had received plasmid DNA fluoresced blue while controls fluoresced green.
  • the ⁇ -lactamase gene of sequence ID 3 was cloned into a Drosophila transformation vector under the control of the glass promotor and injected into wild-type Drosophila embryos. As control, the ⁇ -lactamase gene was inserted in the wrong orientation. Drosophila embryos were germline-transformed using P element-mediated transformation. The transformations and all subsequent fly manipulations were performed using standard techniques [Karess, R.E. and Rubin, G.M., Cell 38 , 135, (1984)]. Omatidia of late stage transformed pupae were transsected and dissociated to single cells.
  • the cells were incubated in buffer with 40 ⁇ molar CCF2/btAMac 2 (compound 34) for 20 minutes, washed and viewed with an epifluorescence microscope (excitation filter 400DF15, emission filter 435 nm long pass). Omatidia cells from flyes transformed with the ⁇ -lactamase gene in the proper orientation fluoresced blue, while omatidia cells containing the gene in the wrong orientation fluoresced green.
  • the compound of this invention can be any of the following compounds. wherein
  • R z and R z1 are independently selected from the group consisting of -C(O)alk, -CH 2 OC(O)alk, -CH 2 SC(O)alk,-CH 2 OC(O)alk, lower acyloxy-alpha-benzyl, and deltabutyrolactonyl; wherein alk is lower alkyl of 1 to 4 carbon atoms.
  • Another example of the compound is:
  • a final example of the compound is:

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